PROJECT SUMMARY ? PROJECT 1 JCPyV infection causes a fatal central nervous system disease, progressive multifocal leukoencephalopathy, PML, for which there are no effective treatments. A thorough understanding of the structure-function relationships of this human pathogen is key for the development of effective therapies against PML in the future. We propose to use structure-function approaches to identify molecules that can bind to the recombinantly produced JCPyV capsid protein VP1 with high affinity (Specific Aim 1). Since the initial hits likely will not be very specific, we will optimize them by conducting binding assays with structurally related compounds using NMR spectroscopy, and verify binding by crystallography as well as in biological assays in collaboration with the Atwood and DiMaio groups (projects #2 and #3). We will furthermore structurally characterize receptor-switching mutants of JCPyV VP1 pentamers and their ligand-binding properties (Specific Aim 2). Our preliminary results show that JCPyV can engage a range of sialylated glycans, but not all of these interactions lead to an infection. We will engineer JCPyV VP1 mutants that selectively use only a single glycan receptor, and we will crystallize the VP1 proteins in complex with the receptor to define specificities and also analyze the corresponding pseudoviruses in collaboration with project #2 and core B for cell binding and entry. Dr. DiMaio (project #3) will determine if viruses targeted to different receptors display different trafficking patterns. In a second part of this aim, we will characterize ligand-binding properties and specificities of JCPyV isolates from PML patients using glycan microarray screening as well as crystallography. Finally, we will perform structure-function analyses of entire JCPyV pseudoviruses (Specific Aim 3). Binding sites for some receptors may lie between VP1 pentamers on the virion surface, and such sites will not be present in the context of the recombinantly expressed pentamers alone. JCPyV pseudoviruses have recently been generated by the Atwood group (project #2), and they faithfully replicate essential functions of the virus in attachment and entry. The JCPyV pseudovirus particles will be analyzed structurally with respect to their ligand binding properties as well as in glycan microarrays.